A cloning of cytidine monophospho-N-acetylneuraminic acid hydroxylase from porcine endothelial cells.

INTRODUCTION:The Hanganutziu-Deicher (H-D) antigen with terminal N-glycolyl neuraminic acid-(NeuGc) is widely distributed in mammalian species including monkeys and apes, but is not found in humans and birds. After the knock out of α1, 3galactosyltransfease, the H-D antigen became a major antigen of the "non-Gal antigen." The expression of NeuGc is controlled by the activity of cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH). In this study, molecular cloning of pig CMAH was performed, as the first step in producing H-D knockout pigs. METHODS:A pig endothelial cell line, MYP30, was used. The DNA sequence of pig CMAH was queried in dbEST (NCBI) using the BLAST program to search for cDNA fragments of pig CMAH, based on an alignment analysis of the mouse CMAH sequence. A polymerase chain reaction experiment was performed and candidate cDNA clones were isolated. To obtain the 5'-end and 3'-end of the open reading frame sequence, a 5'-full RACE Core Set and 3'-full RACE Core Set were used. RESULTS:We cloned and characterized the pig CMAH gene. The ATG is located in exon 4, which corresponds to the mouse gene, and the stop codon is in exon 17. In the case of the 5' site of the gene, exon 3 was identified but exons 1 and 2 are still being investigated. On the other hand, exon 18 was newly identified in the 3' site of the gene. CONCLUSION:The results represent useful information for future clinical xenotransplantation studies.

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